In the past year a method has been developed for separating monomers of chromatin unifilarly substituted with 5-bromodeoxyuridine (HL monomers) from unsubstituted monomers (LL monomers). This technique has been used to study the distribution of the core histones during two cell divisions. No evidence for segregation of core histones was found. In the next year the experiment will be repeated with replicating forks in order to determine whether there is local asymmetric distribution of core histones during DNA replication. Alternative methods for separating two generations of chromatin are underway. HinbIII has been found to distinguish between BrUdRib substituted DNA and unsubstituted DNA. Whether this enzyme can distinguish between substituted and unsubstituted chromatin is now being examined. Conditions have been established for cutting HL chromatin while leaving LL chromatin intact by irradiating briefly with ultraviolet light and then digesting with S1 nuclease. Further work will optimize conditions for separating the two types of chromatin in sucrose gradients. These two methods will be used to study the segregation of non-histone proteins.